The vitamin A metabolite retinoic acid has been recognized as inducing a broad spectrum of biological effects. A variety of structural analogues of retinoic acid have been synthesized that also have been found to be bioactive. Some, such as Retin-A.RTM. (registered trademark of Johnson & Johnson) and Accutane.RTM. (registered trademark of Hoffmann-LaRoche), have found utility as therapeutic agents for the treatment of various pathological conditions. Metabolites of vitamin A and their synthetic analogues are collectively herein called "retinoids". Synthetic retinoids have been found to mimic many of the pharmacological actions of retinoic acid. However, the broad spectrum of pharmacological actions of retinoic acid is not reproduced in full by all bioactive synthetic retinoids.
Medical professionals are interested in the medicinal applications of retinoids. Among their uses approved by the FDA is the treatment of severe forms of acne and psoriasis. Evidence also exists that these compounds can be used to arrest and, to an extent, reverse the effects of skin damage arising from prolonged exposure to the sun. Other evidence indicates that these compounds may be useful in the treatments of a variety of cancers including melanoma, cervical cancer, some forms of leukemia, and basal and squamous cell carcinomas. Retinoids have also been shown to be efficacious in treating premalignant cell lesions, such as oral leukoplakia, and to prevent the occurrence of malignancy.
Retinoids regulate the activity of two distinct intracellular receptor subfamilies; the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). The RAR and RXR subfamilies are divided into six subtypes, based upon their primary sequence homology, their ability to bind to various retinoid analogues, and by their promoter recognition sequence specificity (Mangelsdorf D. J., Umesono K., and Evans R. M. 1994 Retinoid receptors. In: Sporn M. B., Roberts A. B., and Goodman D. S. (eds) The Retinoids: Biology, Chemistry, and Medicine. Raven Press, pp 319-349; Giguere V. 1994, Retinoic acid receptors and cellular retinoid binding proteins: complex interplay in retinoid signalling. Endocrine Reviews 15:in press). The RARs have three subtypes denoted .alpha., .beta., and .gamma.- The RXRs also have three known subtypes, .alpha., .beta., and .gamma..
RARs and RXRs differ in several aspects. First, the RARs and RXRs are divergent in primary structure (e.g., the ligand binding domains of RAR.alpha. and RXR.alpha. have approximately 27% amino acid identity). These structural differences are reflected in the different relative degrees of responsiveness of RARs and RXRs to various vitamin A metabolites and synthetic retinoids. RARs bind to both 9-cis retinoic acid (9cRA) and all-trans retinoic acid (tRA) with equally high affinity, displaying K.sub.d values of 0.2-0.8 nM (Allenby G., et al., 1993, "Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids." Proc Natl Acad Sci USA 90:30-34; Allegretto E. A., et al., 1993, "Transactivation properties of retinoic acid and retinoid X receptors in mammalian cells and yeast: correlation with hormone binding and effects of metabolism." J Biol Chem 268:26625-26633). RXRs bind with high affinity and specificity to 9cRA (Levin A. A., et al., 1992, 9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR.alpha.. Nature 355:359-361; Heyman R. A., et al., 1992, 9-cis retinoic acid is a high affinity ligand for the retinoid X receptor. Cell 68:397-406) with K.sub.d values of 1-2 nM (Allegretto EA, et al., 1993, "Transactivation properties of retinoic acid and retinoid X receptors in mammalian cells and yeast: correlation with hormone binding and effects of metabolism." J Biol Chem 268:26625-26633), but do not bind to tRA (IC.sub.50 &gt;50,000 nM versus tritiated 9 cRA (Allenby G., et al., 1993, "Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids." Proc Natl Acad Sci USA 90:30-34; Allegretto E. A., et al , 1993, "Transactivation properties of retinoic acid and retinoid X receptors in mammalian cells and yeast: correlation with hormone binding and effects of metabolism." J Biol Chem 268:26625-26633)). In addition, distinctly different patterns of tissue distribution are seen for RARs and RXRs. For example, in contrast to the RARs, which are not expressed at high levels in the visceral tissues, RXR.alpha. mRNA has been shown to be most abundant in the liver, kidney, lung, muscle and intestine. Furthermore, RARs and RXRs have different target gene specificity. For example, response elements in cellular retinol binding protein type II (CRBPII) and apolipoprotein AI genes confer responsiveness to RXR, but not to RAR. RAR has also been shown to repress RXR-mediated activation through the CRBPII RXR response element (Mangelsdorf et al., Cell, 66:555-61 (1991)).
Gaub, M. P., et al., Proc. Natl. Acad. Sci. USA., 86:3089-3093, (1989) used synthetic peptides to generate anti-RAR.alpha. and anti-RAR.beta. antisera and used these antisera to identify RAR.alpha. and RAR.beta. expressed in HL-60 cells.
Rochette-Egly, C., et al., J. Cell Bio., 115(2):535-545, (1991) used synthetic peptides to generate anti-RAR.gamma. 1 antibodies which were used to detect mRAR.gamma. 1 and mRAR.gamma. 2.
Gaub, M. P., et al., Experi. Cell Res., 201(2)335-346, (1992) used synthetic peptides or fusion proteins to generate human and mouse RARe 1 antibodies.
Rochette-Egly, C., et al., Mole. Endocri., 6(12):2197-2209, (1992) used synthetic peptides or fusion proteins to generate human and mouse RAR.beta. antibodies.
Heyman, R. A., et al., Cell, 68:397-406, 1992, used an hRXR.alpha. antiserum in Western blot analysis. The antibody "was a rabbit polyclonal serum raised against a synthetic peptide corresponding to amino acids 214-229 (DRNENEVESTSSANED) of hRXR.alpha.." Id. at 405.
Marks, M. S., et al., Mole. Endocrio., 9(2):219, (1992) made an antipeptide antibody that reacts with the recombinant H-2RIIBP and used the antibody in Western blot analysis.